Recombinant Mouse EphB6

Using Recombinant Mouse EphB6 in research applications is valuable for studying T cell activation, immune signaling, and cancer biology, as EphB6 is a key modulator of immune cell function and tumor progression.

Key scientific reasons to use recombinant Mouse EphB6:

• T Cell Costimulation and Activation: EphB6 acts as a novel costimulatory molecule for T cells. Crosslinking EphB6 with its ligand or antibodies enhances T cell proliferation and cytokine production, lowering the activation threshold and increasing responsiveness to TCR stimulation. Recombinant EphB6 enables controlled in vitro studies of these mechanisms.

• Dissecting Immune Signaling Pathways: EphB6 is involved in downstream signaling events following TCR activation, including ZAP-70 activation, LAT phosphorylation, and MAPK pathway engagement. Recombinant protein allows for mechanistic studies, such as ligand-receptor binding assays and signaling pathway analysis.

• Functional Studies in Disease Models: EphB6 knockout mice exhibit compromised T cell responses and altered susceptibility to autoimmune and inflammatory diseases, highlighting its role in immune regulation. Recombinant EphB6 can be used to rescue or modulate these phenotypes in vitro or in vivo.

• Cancer Research Applications: EphB6 expression is linked to tumor cell behavior, including proliferation, survival, and resistance to therapy in certain leukemias and solid tumors. Recombinant EphB6 can be used to probe its role in tumor cell signaling, cell-cell interactions, and as a potential therapeutic target.

• Ligand Binding and Receptor Interaction Studies: Recombinant EphB6 is essential for binding assays with ephrin ligands (such as ephrinB2) and for mapping receptor-receptor interactions, including those with other Eph family members (e.g., EphA2, EphB2).

• Antibody and Drug Screening: Recombinant protein is required for developing and validating antibodies or small molecules targeting EphB6, which may have diagnostic or therapeutic potential.

Additional considerations:

• Recombinant proteins provide a consistent, defined reagent for reproducible experiments, avoiding variability seen with endogenous protein sources.
• Mouse EphB6 is highly homologous to human EphB6, making it suitable for translational studies and cross-species comparisons.

In summary, recombinant Mouse EphB6 is a critical tool for dissecting its immunological and oncological functions, enabling precise experimental manipulation and mechanistic insight into its biological roles.

You can use recombinant Mouse EphB6 as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and it is compatible with your assay's antibody pair and detection system.

Key considerations:

• Purity and Quantification: The recombinant protein should be highly purified (typically >90% by SDS-PAGE) and its concentration must be precisely measured, ideally by absorbance at 280 nm or another validated method.
• Standard Curve Preparation: Prepare a dilution series of the recombinant EphB6 in the same buffer or matrix as your samples to generate a standard curve. This is a standard approach for quantitative ELISA.
• Antibody Compatibility: Ensure that the antibodies used in your ELISA recognize the recombinant form of EphB6. Available data indicate that commonly used anti-EphB6 antibodies detect recombinant mouse EphB6 in direct ELISA formats.
• Validation: It is essential to validate that the recombinant EphB6 standard produces a dose-response curve parallel to that of endogenous EphB6 in your sample matrix. This confirms that the recombinant protein is immunologically equivalent to the native protein in your assay context.
• Documentation: Some commercial ELISA kits provide their own validated standards and recommend against substituting other proteins, as this may affect assay accuracy and reproducibility. If you are developing a custom assay, using a well-characterized recombinant standard is common practice.

Limitations:

• If your recombinant EphB6 is a fusion protein (e.g., Fc chimera), ensure that the fusion does not interfere with antibody binding or assay performance.
• If the ELISA kit is designed for a specific standard, substituting with another recombinant protein may require additional validation to ensure accuracy.

In summary, recombinant Mouse EphB6 can be used as a standard for ELISA quantification if it is well-characterized, pure, and validated for your specific assay system. Always confirm parallelism and antibody compatibility before relying on it for quantitative results.

Recombinant Mouse EphB6 has been validated for several key applications in published research, primarily in the context of biochemical, structural, and functional studies. The main applications include:

1. Protein Expression and Purification
   Recombinant Mouse EphB6 has been expressed and purified from mammalian (HEK293) and insect (Spodoptera frugiperda 21) cell systems for use in downstream assays. These purified proteins are used for structural and functional characterization.

2. Thermal Shift Assays
   Recombinant EphB6 protein is used in thermal shift assays to study the binding of nucleotides and kinase inhibitors, helping to identify potential pharmacological targets.

3. In Vitro Kinase Activity Assays
   Recombinant EphB6 is used in kinase assays to assess phosphorylation activity, often in combination with other Eph receptors (e.g., EphB4), and to study downstream signaling events.

4. Surface Plasmon Resonance (SPR) Experiments
   Recombinant EphB6 is employed in SPR studies to measure interactions between phosphorylated EphB6 and SH2 domain-containing signaling proteins, such as Abl, Src, and Vav3.

5. Structural Studies
   Recombinant EphB6 ectodomain and intracellular domains have been used in structural biology approaches, including small-angle X-ray scattering (SAXS) and cross-linking mass spectrometry, to investigate domain flexibility and inter-domain interactions.

6. Signaling Pathway Analysis
   Recombinant EphB6 is used to study its role in cellular signaling, particularly in T-cell responses and cancer biology, including modulation of Akt signaling and regulation of cell proliferation.

These applications highlight the utility of recombinant Mouse EphB6 in both basic research and drug discovery, especially in the context of immune regulation and oncology.

To reconstitute and prepare Recombinant Mouse EphB6 protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) at a concentration of 100 μg/mL. This concentration is commonly used for Fc chimera formats and provides a suitable starting point for most cell-based assays.

Essential steps and considerations:

• Reconstitution:

  • Add sterile PBS directly to the lyophilized protein to achieve the desired concentration (e.g., 100 μg/mL).
  • Gently mix by pipetting up and down or by rocking the tube to ensure complete dissolution.
  • Avoid vigorous vortexing, which may denature sensitive proteins.

• Sterility:

  • Use sterile technique throughout to prevent contamination.
  • If required, filter the reconstituted solution through a 0.22 μm sterile filter.

• Aliquoting and Storage:

  • Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles, which can degrade protein activity.
  • Store aliquots at −80 °C for long-term preservation.
  • Thaw aliquots on ice before use and keep them cold during handling.

• Buffer Compatibility:

  • PBS is generally suitable for cell culture applications, but confirm compatibility with your specific cell type and assay.
  • If the protein is sensitive to oxidation, consider adding a reducing agent such as 0.5 mM TCEP (tris(2-carboxyethyl)phosphine) for stability, as used in some purification protocols.

• Working Concentration:

  • For cell stimulation or binding assays, typical working concentrations range from 1–10 μg/mL, but optimal dosing should be determined empirically for your experimental system.

• Application:

  • Add the reconstituted protein directly to cell culture media or coat onto tissue culture plates, depending on your experimental design.
  • For plate coating, incubate wells with the protein solution (e.g., 5–10 μg/mL) for 2 hours at 37 °C, then wash before adding cells.

General best practices for recombinant protein handling:

• Always consult the specific product datasheet for recommended reconstitution buffers and concentrations.
• Avoid repeated freeze-thaw cycles.
• Confirm protein integrity and activity by SDS-PAGE or functional assay before use in critical experiments.

If your recombinant EphB6 is not an Fc chimera or has a different format, adjust the buffer and concentration according to the manufacturer’s instructions or published protocols. For most cell culture applications, PBS at neutral pH is appropriate unless otherwise specified.